Multi drug-resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis (TB) is increasing worldwide. M. tuberculosis does not naturally contain plasmids and almost all cases of clinical drug-resistance are caused by single-nucleotide polymorphisms (SNPs) or small insertions/deletions in relevant genes. In the case of Rifampicin-resistance, 95-98% of Rifampicin-resistant clinical strains have mutations in the eighty base pair Rifampicin Resistance Determining Region (RRDR) of the M. tuberculosis RNA polymerase beta (rpoB) gene. PCR and probe-based molecular genotyping assays can be used to detect these resistance-inducing mutations. Such genotypic assays are potentially more rapid than labor intensive culture-based drug susceptibility tests.
The Xpert MTB/RIF assay (Cepheid, Sunnyvale, Calif.) is one example of a genotypic test that is being increasingly used to screen for Rifampicin-resistance. However, the single use cartridge design of the Xpert assay limits its use for laboratory-based high-throughput testing. Several widely used reverse blot hybridization assays such as the INNO-LIPA Rif.TB assay (Innogenetics, Belgium) and the MTBDRplus (Hain, Germany) assay are available for laboratory-based Rifampicin-resistance screening; however, these assays are complicated by their open hybridization format. Open hybridization systems require a relatively cumbersome work process including rigorous physical separation of different work areas due to the risk of handling open PCR amplicons in a molecular diagnostic laboratory (Albert, H., et. al. 2010. BMC Infect Dis 10:41). Open systems also require a relatively large number of probes to test for relevant resistance-associated mutations. This requirement complicates assay chemistry and hybridization parameters. There is a need for a rapid single-PCR assay to identify Rifampicin-resistant M. tuberculosis with RRDR mutations, that has high sensitivity and specificity.